Since the introduction of genetically modified (GM) crops, there has been a rapid adoption of the technology. The International Service for the Acquisition of Agri-Biotech Application (ISAAA) reported that the global acreage of GM crops reached a record 160 million hectares in 29 countries in 2011. United States of America, Brazil, Argentina, India, Canada and China represent the six largest countries for planting GM crops. Currently, the major GM field crops are soybean, corn, cotton and canola. Minor GM crops include papaya, sugarbeet, squash, potato and alfalfa. Foreign gene expression cassettes are typically delivered into plants by Agrobacterium-mediated transformation and biolistic bombardment transformation. Agrobacterium tumefaciens has been widely used for introducing genes into plants for purposes of basic research as well as for the generation of commercial transgenic crops. Agrobacterium-mediated transformation of plants can result in DNA sequences outside the T-DNA region, such as vector backbone, to integrate into the plant genome (Kononov et al. Plant J 11: 945-957 (1997); Wenck et al. Plant Mol Biol 34: 913-922 (1997); Shou et al. Mol Breed 13: 201-208 (2004)). It is common practice for vector backbone sequences to be removed before transforming foreign DNA into commercial crops using the particle bombardment transformation approach. Further, it has been reported that biolistic delivery of a linear construct with no vector backbone has been shown to produce a high percentage of events with intact single copy insertions (Fu et al. Transgenic Res. 9:11-19 (2000)).
The current approach to removing unwanted vector backbone sequences, i.e., the bacterial replication DNA region, prior to plant transformation involves restriction enzyme digestion, preparative gel electrophoresis, then confirmation by analytical gel electrophoresis against reference standards. However, the current approach has disadvantages: each construct must be checked for available sites for E. coli plasmid backbone removal for every construct. To estimate E. coli DNA in the preps, the DNA preparations need to be serially diluted and the diluted DNAs back transformed into E. coli to check for unwanted circular DNA. A calculation using this method is not accurate. Further, the process is inefficient and a large amount of the DNA intended for transformation is wasted. Thus, this method is complicated, time consuming, has low throughput and is inaccurate. Additionally, no assay is available for detection of E. coli backbone containing plasmid DNA elements such as ColE region because (1) Taq DNA polymerase used in the detection assays is contaminated with E. coli DNA and/or TAQ expression plasmid DNA each providing a potential source of plasmid DNA elements such as ColE region which interferes with measurement of ColE in the DNA sample intend for use in transformation.
The present invention overcomes the shortcomings in the art by providing methods for determining the presence of vector backbone in purified nucleic acid preparations that are not only rapid but have greater accuracy.